Nitric oxide (NO), which has demonstrated plasticity regarding processes of learning and memory, has revealed its specific function in memory formation for the honeybee. The predominant amount of NO synthase (NOS) activity has been shown to participate in the processing of olfactory information of a honeybee. A honeybee's antenna (*- Application of appetitive stimuli to the antennae of honeybees elicits extension of the proboscis (PER)) lobes are the primary centers of olfactory processing that exhibit highest NOS activity, which takes place in the brain. The lip of the mushroom body, which is involved in this process, calyces and the lateral protocerebral lobe receives input from the antennal lobs via a median and a lateral pathway; this indicates strong labeling. Neuropils of the central brain exhibited intermediate labeling.
The effects of NOS activity on honeybees were revealed injecting a hemoglobin assay. The hemoglobin assay is able to detect NO and other substances (CO, etc.), therefore, these assays reveal at least two signals. By adding NOS inhibitors to the assay mixture, the first signal can be inhibited, but the second signal cannot be inhibited. The second signal includes enzymes other than NOS. Total NOS activity (*- NOS activity: defined as that part of the signal which is sensitive to NOS inhibitors added to the assay mixture) is divided into a major Ca2+ and a minor Ca2+ independent NOS activity which suggested that the existence of at least two NOS isoforms in the honeybee brain.
Applying only 1 (l of 100 (l N-nitro-L-arginine or N-nitro-L-arginine methyl ester (L-NAME) 20 minutes inhibits total NOS by 80% (*-show figure 2A). Maximal inhibition of NOS was attained at an approximate concentration of 1 (M NOS inhibitor which was calculated from the injected volume and the approximate body weight of 120 mg of the honeybee. Phosphate-buffered saline (PBS) or inactive isomer N-nitro-D-arginine methyl ester (D-NAME) served as injected controls showed no effect on NOS activity. NOS inhibition was shown to have a maximal effect around 20 minutes after injection of the competitive inhibitor NOArg (show Figure 2B). L-NAME injection at the same concentration leads to a similar transient NOS inhibition.
In this experiment, a honeybee's proboscis extension response (PER) was be conditioned by pairing an odor stimulus with a sucrose reward (Menzel, 1985, 1990). Throughout condition trials, the data collected suggested that the number of conditioning trials applied to the honeybee induces different memories that exhibit different properties. A single trial conditioning (*- Blocking of NOS during conditioning shows distinct effects on the conditioned PER tested at 24 hours, depending on the number of conditioning trials applied to the animal.) (tested 30 minutes after conditioning) leads to a medium-term memory that lasts for a couple of hours, while multiple trial conditioning induced a long-term memory in the...